Polistes dominula

Nineteen post-emergence nests were collected in the vicinity of Leuven (Belgium, 50°53′ N, 4°42′ E) between the end of June and the end of August 2018. Subsequently, all adult individuals were removed, and the nests were glued into wooden boxes (20 × 20 × 13 cm), of which one side was made of Perspex. The nests were kept inside the laboratory under a controlled temperature of 28°C and a 12:12 day/night cycle. Every other day the box was checked for newly emerged individuals. Any newly emerged individuals were treated once with either 2 µL of a methoprene solution (20 µg/µL in acetone, Sigma–Aldrich), 2 µL of a precocene solution (20 µg/µL in acetone, Sigma–Aldrich) or acetone solvent (control group, VWR chemicals), applied topically onto the abdomen. The doses of methoprene and precocene chosen were based on comparable experiments carried out in related species (Izzo et al. 2010; Oliveira et al. 2017). After treatment, the individuals were paint marked according to their treatment using acrylic paint and placed in the nest where they were originated from. Hence, each nest contained individuals of the three groups: treated once with either methoprene, precocene, or acetone sham-treated control. The maximum number of individuals allowed in one box was 20 adults. Whenever this limit was reached, a new box without comb was set up for the remaining individuals. The wasps were fed using mealworms (Tenebrio molitor) and sugar water ad libitum. Ten to 12 days after their initial treatment, the individuals were frozen at −20°C to be dissected, in order to assess their ovary activation and to determine their CHC profiles.